Fusokines Involving Cytokines with Strongly Reduced Receptor Binding Affinities

ABSTRACT

The present invention relates to a fusion protein comprising at least two cytokines, of which at least one is a modified cytokine with a strongly reduced binding affinity to its receptor, or to one of its receptors. Preferably, both cytokines are connected by a linker, preferably a GGS linker. The invention relates further to said fusion protein for use in treatment of diseases.

The present invention relates to a fusion protein comprising at leasttwo cytokines, of which at least one is a modified cytokine with astrongly reduced binding affinity to its receptor, or to one of itsreceptors. Preferably, both cytokines are connected by a linker,preferably a GGS linker. The invention relates further to said fusionprotein for use in treatment of diseases.

Cytokines are small secreted or membrane-bound proteins which play acrucial role in intercellular communication. Cytokine binding to itscognate receptor complex triggers a cascade of intracellular signalingevents that enables the cell to sense and respond to its surroundingsaccording to the needs of the cell, tissue and organ of which it is partof. They are characteristically pleiotropic, meaning that they provoke abroad range of responses depending on the nature and the developmentalstate of the target cell. Moreover, some of them are highly redundant asseveral cytokines have overlapping activities, which enable them tofunctionally compensate for mutual loss. Cytokine activities can beautocrine, paracrine or endocrine causing a faint boundary between thedesignated term cytokine, peptide hormone and growth factor.

Six different structural classes of cytokines are known: the α-helicalbundle cytokines which comprises most interleukins, colony stimulatingfactors and hormones like growth hormone and leptin (Nicola and Hilton,1998), the trimeric tumor necrosis factor (TNF) family (Idriss andNaismith, 2000), the cysteine knot growth factors (Sun and Davies,1995), the β-trefoil fold group that includes the interleukin-1 family(Murzin et al., 1992), the interleukin 17 (IL-17) family (Gaffen, 2011),and the chemokines (Nomiyama et al., 2013).

Several cytokines have found important clinical applications. Examplesinclude erythropoietin (Epo), granulocyte colony-stimulating factor(G-CSF), interferons α2 and -β, and growth hormone. Conversely, often asa consequence of their pro-inflammatory nature, antagonizing selectedcytokines also finds specific medical applications. Prime examples hereare the strategies to block TNFα activity to combat autoimmune diseasessuch as rheumatoid arthritis. Because of these successes, strategies tooptimize cytokine activities in the clinic are being explored. Theseinclude optimized half-life, reduced immunogenicity, targeted deliveryto specific cell types and genetic fusions of two cytokines, so-calledfusokines.

Fusokines are artificial combinations of two different cytokines whichare genetically linked using a linker sequence. The first example of afusokine is pIXY321 or pixykine which is a fusion protein ofgranulocyte-macrophage colony-stimulating factor (GMCSF) and IL-3(Donahue et al., 1988) that showed superior hematopoietic and immuneeffects compared to either cytokine alone. This effect could beexplained by enhanced binding to their respective receptor complexes. Ofnote, both receptors share the signaling βc subunit, precludingsynergistic effects at the signal transduction level. In a Phase IIIclinical trial, pIXY321 did not show superior properties when comparedto GM-CSF alone (O'Shaughnessy et al., 1996). GM-CSF-based fusokineswith cytokines of the IL-2 family were explored as well. These cytokinesall signal through receptor complexes comprising the γc subunit.Examples of such fusokines with GM-CSF include IL-2 (Stagg et al.,2004), IL-15 (Rafei et al., 2007) and IL-21 (Williams et al., 2010a),aka as GIFT2, -15 and -21. Synergistic effects could be expected both atthe signaling level (i.e. synergistic effects within a target cell) andcellular level (i.e. synergistic effects between different target celltypes). For example, GIFT2 induced more potent activation of NK cellscompared to the combination of the unfused cytokines (Penafuerte et al.,2009) and GIFT15 induced an unanticipated, potent immune-suppressiveB-cell population (Rafei et al., 2009a). Likewise, GIFT21 exertedunexpected proinflammatory effects on monocytic cells (Williams et al,2010b). Another example of a fusokine that combines α-helical cytokinesis IL-2/IL-12 (Gillies et al., 2002; Jahn et al, 2012).

Another class of fusokines combines cytokines from different structuralfamilies. Examples include the fusion of IL-18 (a member of the IL-1cytokine family) and IL-2 (Acres et al., 2005) and the fusion betweenIL-18 and EGF (epidermal growth factor). Since overexpression of theEGFR is often observed on certain tumor cell types, the latter fusokineoffers the possibility to target the IL-18 activity to EGFR+ tumor cells(Lu et al., 2008). Fusions between α-helical bundle cytokines andchemokines were also explored in greater detail. Chemokines often actusing concentration gradients to steer migration of immune cells tosites of infection and inflammation. Many chemokine receptors display arestricted expression pattern allowing targeting to selected (immune)cells. Moreover, signaling via the serpentine, G-protein coupledchemokine receptors is fundamentally different from pathways activatedby the α-helical bundle cytokine receptor complexes and synergeticpositive and negative cross-talk mechanisms could be expected. Of note,designed N-terminally truncated versions of chemokines can retain theirreceptor binding properties but display antagonistic behavior. Anexample is a fusokine between GM-CSF and a N-terminally truncated CCL2lacking the first 5 N-terminal amino-acids, aka GMME1 (Rafei et al.,2009b). This fusokine induced the apoptosis of inflammatory CCR2+ cellsand mice treated with GMME1 displayed reduced experimentally-inducedautoimmune disease scores including EAE and CIA for multiple sclerosis(Rafei et al., 2009b) and rheumatoid arthritis (Rafei et al., 2009c),respectively. Likewise, this fusokine induced apoptosis of CCR2+ tumorcells (Rafei et al., 2011).

However, fusions between a wild-type cytokine and a mutant cytokine withstrongly reduced affinity for its cognate receptor complex were notexplored before. The advantage of this approach is that the possiblesystemic toxicity of the wild type cytokine is eliminated. Surprisingly,we found that such fusokines allow cell-specific targeting of cytokineactivities whereby such mutant cytokine can regain its activity on thetargeted cells, without the negative effect of wild type cytokines. Thegeneral applicability of the principle has been demonstrated using threefusokines each composed of two cytokines from structurally differentcytokine classes, as exemplified below.

XCL1/IFNα2-Mutant

XCL1 is a 93 amino acids chemokine secreted by CD8⁺ T cells, Th1cell-polarized CD4⁺ T cells and NK cells. It interacts with XCR1, achemokine receptor exclusively expressed by dendritic cells. In mice,XCR1 is expressed in the large majority of splenic CD11c⁺ CD8α⁺dendritic cells whereas only a very minor subset of CD8α⁻ dendriticcells expresses this receptor (Dorner et al. 2009). XCR1 is a conservedselective marker of mammalian cells (including human cells) homologousto mouse CD8α⁺ dendritic cells (Crozat et al. 2010). Interestingly ithas been shown that the action of type I interferon (IFNα/β) on thisdendritic cell subset is critical for the innate immune recognition of agrowing tumor in mice (Fuertes et al. 2011).

Systemic IFNα therapy has considerable toxicity, including side effectssuch as severe fatigue, fever, chills, depression, thyroid dysfunction,retinal disease, hair loss, dry skin, rash, itching and bone marrowsuppression. It would thus be highly worthwhile to target IFN activitytoward only the cellular population which should be treated with IFN.For application in antitumor therapies, targeting the population ofXCR1-expressing dendritic cells is highly desirable since these cellsare specialized in antigen cross-presentation (Bachem et al. 2012). Manyexperimental data suggest that the XCR1-expressing dendritic cellpopulation represents the key cellular population which must react withtype I IFN in the tumor microenvironment in order to initiate the immuneresponses which ultimately will allow tumor destruction and immunization(Gajewski et al. 2012).

The human IFNα2-Q124R mutant has a high affinity for the murine IFNAR1chain and a low affinity for the murine IFNAR2 chain (Weber et al.,1987). It displays a very low activity on murine cells and hencerepresents a prototype of an engineered type I IFN subtype suitable totarget IFN activity on selected mouse cells (PCT/EP2013/050787).

CCL20/IL1β

The CC chemokine CCL20, also known as liver and activation-regulatedchemokine (LARC), macrophage inflammatory protein-3α (MIP-3α) orExodus-1 is a 96 AA protein that is predominantly expressed in liver andlymphoid tissue (Hieshima et al., 1997). Upon secretion, CCL20 exertsits activity by binding to the CC chemokine receptor 6 (CCR6), whichbelongs to the G-protein coupled receptor (GPCR) 1 family (Baba et al.,1997). CCR6 expression is reported on different leukocyte subsets but isbest documented for the Th17 cell population (Singh et al., 2008).Normal Th17 function is indispensable for protective immunity against arange of pathogens, including Mycobacterium tuberculosis (Khader et al.,2007), Klebsiella pneumoniae (Ye et al., 2001) and Bordetella pertussis(Higgins et al., 2006).

Potentiating effects of IL-1β on the expansion and differentiation ofdifferent T cell subsets, in particular Th17 cells (Sutton et al., 2006;Acosta-Rodriguez et al., 2007; Dunne et al., 2010; Shaw et al., 2012)have been firmly established. Among T cell subsets, Th17 cells expressthe highest levels of the IL-1R and IL-1 plays an important role in Th17priming. Controlled agonistic IL-1 activity could therefore haveapplications in different physiological/pathological processes, whereimmunostimulatory effects would be desirable. One of the main concernsregarding the use of IL-1 in immunostimulatory therapies is however itssevere toxicity when administered systemically. Thus, when IL-1 actioncould be confined to a selected cellular population, the toxicity issuemight be resolved, which opens up therapeutic perspectives, e.g. for theuse as a T-cell adjuvant to enhance the response to weak vaccines(Ben-Sasson et al., 2011). To specifically target IL-1 mutants to theTh17 cell population, IL-1 variants are used that consist of mutant IL-1fused to a CCL20 targeting moiety. Because activation will be confinedto CCR6-expressing cells (ie Th17 cells) only, no major systemictoxicity is expected.

TNFα/Leptin Mutant

TNFα is a cytokine with a wide range of biological activities includingcytotoxicity, regulation of immune cells and mediation of inflammatoryresponses. It is a self-assembling, non-covalently bound, homotrimerictype II transmembrane protein of 233 amino acids. TNFα is active as amembrane-bound as well as a soluble protein, released from the cellmembrane after proteolytic cleavage of the 76 aminoterminal amino acids(presequence) by TNFα converting enzyme (TACE, also called ADAM17). Itsignals through 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75),both transmembrane glycoproteins with a cystein-rich motif in theligand-binding extracellular domain. Despite the extracellular homology,they have distinct intracellular domains and therefore signal differentTNF activities (Hehlgans & Pfeffer, 2005). We generated a single chainvariant (scTNF) that consists of three TNF monomers coupled viaGGGGS-linkers as described before by Boschert et al., 2010.

Leptin is a 16 kDa adipocytic cytokine involved in a multitude ofbiological processes, including immunity, reproduction, linear growth,glucose homeostasis, bone metabolism and fat oxidation, but is bestknown for its dramatic effect as a satiety signal (Halaas et al., 1995).Because of its effect on immune cells, leptin is also implicated inseveral auto-immune diseases (likuni et al., 2008). Selective targetingof leptin activity may be beneficial for both metabolic and immune- orinflammation-related disorders.

A first aspect of the invention is a fusion protein, comprising at leasttwo cytokines, of which at least one cytokine is a modified cytokinethat shows a strongly reduced binding activity towards its receptor, ortowards at least one of its receptors, if binding on different receptorsis possible. A reduced binding affinity, as used here, means that theaffinity is less than 50%, preferably less than 40%, more preferablyless than 30%, more preferably more than 25%, more preferably less than20%, more preferably less than 15%, more preferably less than 10%, morepreferably less than 5%, most preferably less than 1% of the wild typecytokine. “Wild type cytokine” as used here, means the cytokine as itoccurs in nature, in the host organism. The modification of the cytokineresulting in a reduction in binding affinity can be a modification thatdecreases the activity of the normal wild type cytokine, or it can be amodification that increases the affinity of a homologous, non-endogenouscytokine (such as, but not limited to a mouse cytokine, binding to ahuman cytokine receptor). Modifications can be any modification reducingor increasing the activity, known to the person skilled in the art,including but not limited to chemical and/or enzymatic modificationssuch as pegylation and glycosylation, fusion to other proteins andmutations. Preferably, the cytokine with reduced binding affinity to thereceptor is a mutant cytokine. The mutation may be any mutation known tothe person skilled in the art, including deletions, insertions,truncations or point mutations. Preferably, said mutation is a pointmutation or a combination of point mutations. The affinity can bemeasured with any method known to the person skilled in the art. As anon-limiting example, the affinity of the ligand towards the receptorcan be measured by Scatchard plot analysis and computer-fitting ofbinding data (e.g. Scatchard, 1949) or by reflectometric interferencespectroscopy under flow through conditions, as described by Brecht etal. (1993).

Alternatively, the reduced binding activity can be measured as reductionof the biological activity of the mutant ligand compared to the wildtype ligand. In a preferred embodiment, said biological activity ismeasured in vitro, using a reporter assay. Such reporter assays dependupon the cytokine receptor system used, and are known to the personskilled in the art. As a non-limiting example, an IFN-γ reporter assayis described by Bono et al (1989) together with the Scatchard analysis.Preferably the biological activity of the mutant is less than 50%,preferably less than 40%, more preferably less than 30%, more preferablymore than 25%, more preferably less than 20%, more preferably less than15%, more preferably less than 10%, more preferably less than 5%, mostpreferably less than 1% of the wild type cytokine

The modified cytokine is fused to another cytokine, modified or not.Preferably, both cytokines are fused using a linker sequence, preferablya GGS linker, comprising one or more GGS repeats. The modified cytokinemay be placed in the aminoterminal part of the molecule, or in thecarboxyteminal part; the fusion protein may further comprise otherdomains such as, but not limited to a tag sequence, a signal sequence,another cytokine or an antibody.

A cytokine as used here may be any cytokine known to the person skilledin the art, including, but not limited to cytokines of the α-helicalbundle cytokine family, the trimeric tumor necrosis factor (TNF) family(Idriss and Naismith, 2000), the cysteine knot growth factors (Sun andDavies, 1995), the β-trefoil fold group that includes the interleukin-1family (Murzin et al., 1992), the interleukin 17 (IL-17) family (Gaffen,2011), and the chemokines (Nomiyama et al., 2013). In case of a memberof the trimeric TNF family, preferably a single chain version is used.Such single chain cytokines are known to the person skilled in the art,and are described, amongst others, by Krippner-Heidenrich et al. (2008)

In one preferred embodiment, said fusion protein is a fusion betweenXCL1 and a IFNα2-mutant, preferably a Q124R mutant. In another preferredembodiment, said fusion is a fusion between CCL20 and an IL1β mutant.Preferably, said IL1β mutant is a Q148G mutant. In still anotherpreferred embodiment, said fusion is a fusion between TNFα and a leptinmutant. Preferably, said leptin mutant is a selected from the groupconsisting of L86S and L86N.

Another aspect of the invention is a fusion protein according to theinvention for use as a medicament. In one preferred embodiment it is afusion protein according to the invention for use in treatment ofcancer. In another preferred embodiment, it is a fusion proteinaccording to the invention for use in modulation of the immune response.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the structural elements in theXCL1/IFNα2-Q124R fusion protein.

FIG. 2: Selective activity of the XCL1/IFNα2-Q124R fusion protein onXCR1 expressing cells.

STAT1 Y701 phosphorylation is measured in response to IFNα/β orXCL1/IFNα2-Q124R fusion protein in different mouse splenocyte subsets,characterized by the expression of CD11c and CD8α. First column: CD11c⁻CD8α⁻ subset; second column: CD11c⁻ CD8α⁻ subset; third column:CD11c^(medium) CD8α⁻ subset; fourth column: CD11c^(high) CD8α⁺ subset;fifth column: CD11c^(high) CD8α⁻ subset.

FIG. 3: Schematic representation of the structural elements in theIL-1β-mutant/CCL20 fusion proteins.

FIG. 4: Selective activity of the IL-1β-mutant/CCL20 fusion proteins onCCR6 expressing cells.

(A) induction of NFκB activity by wild type and 5 different IL-1βmutants, fused to CCL20.

(B) concentration dependency of the induction of the NFκB activity bywild type and IL-1β Q148G mutant/CCL20 fusion proteins, in mocktransfected cells, or cells transfected with CCR6.

(C) induction of the NFκB activity by wild type and IL-1β Q148Gmutant/CCL20 fusion proteins (12.5 ng/ml), in mock transfected cells, orcells transfected with CCR6 as compared with induction by vehicle.

FIG. 5: Schematic representation of the structural elements in thescTNFα/Leptin-mutant fusion proteins.

FIG. 6: Selective activity of the scTNFα/Leptin mutant fusion proteinson leptin receptor expressing cells.

Leptin-dependent growth induced by indicated concentrations ofscTNF-targeted WT or mutant leptin is measured by the XTT assay inBa/F3-mLR cells (panel A) or Ba/F3-mLR-TNFR1ΔCyt cells (panel B).

FIG. 7: In vivo targeting of IFN activity on mouse spleen cellsexpressing XCR1.

C57Bl/6 mice were injected iv with the indicated amount ofXCL1-IFNα2-Q124R or with 1 000 000 units of natural murine IFNα/β orPBS. After 45 min, spleen cells were analyzed by FACS for CD11c and CD8aexpression (first panel) and for P-STAT1 (further panels) in thefollowing cell population: CD11c− CD8α− (line 1), CD11c− CD8α+(line 2),CD11c+CD8α+(line 3), CD11c+CD8α− (line 4).

EXAMPLES

Materials & Methods to the Examples

Cloning and Production of the Fusokines

Cloning of the XCL1/IFNα2-Q124R Fusion Protein.

The XCL1 open reading frame was synthesized by PCR from theXCL1-encoding plasmid MR200473 (Origen Inc.), using the Expand HighFidelity PCR system (Roche Diagnostics) and the following primers:

Forward: 5′GGGGGGGAATTCATGAGACTTCTCCTCCTGAC3′ Reverse:5′GGGGGGTCCGGAGGCCCAGTCAGGGTTATCGCTG3′

The PCR product was digested by EcoRI and BspEI and substituted to theEcoRI-BspEI fragment which encodes the nanobody in the pMET7SIgK-HA-1R59B-His-PAS-ybbr-IFNA2-Q124R vector (PCT/EP2013/050787).

Production of the XCL1/IFNα2-Q124R Fusion Protein.

Hek 293T cells were transfected with the protein fusion construct usingthe standard lipofectamin method (Invitrogen). 48 hours after thetransfection culture medium were harvested and stored at −20° C. The IFNactivity was assayed on the human HL116 and the murine LL171 cell linesas described (Uzé et al. J. Mol. Biol. 1994) using the purified NanobodyGFP-IFNα2-Q124R preparation (described in PCT/EP2013/050787) as astandard.

Cloning of IL-1p/CCL20 Fusion Proteins.

A codon-optimized sequence encoding the mature human IL-1β/CCL20 fusionprotein was generated via gene synthesis (Invitrogen Gene Art). Briefly,a sequence was synthesized in which the mature human IL-1β protein,preceded by the SigK leader peptide, and equipped with an N-terminal HA,was fused at its C-terminus to a 13xGGS linker sequence, followed by thesequence for mature human CCL20 with a C-terminal HIS tag (FIG. 3).

IL-1β mutants expected to have reduced binding affinity for the IL-1βwere selected based on literature and analysis of published crystalstructures of human IL-1β complexed with its receptor. Mutations in thehIL-1β moiety were created via site-directed mutagenesis (QuickChange,Stratagene) using the mutagenesis primers as indicated in the table:

Fw primer Rev primer R120G GCGGCAGCGCCCCTGTCGGA GCAGGGTGCAGTTCAAGCTTCCGAAGCTTGAACTGCACCCTGC CAGGGGCGCTGCCGC Q131G CTGCGGGACAGCCAGGGGAACGCTCATGACCAGGCTCTTCCCCT GAGCCTGGTCATGAGCG GGCTGTCCCGCAG H146ACGAGCTGAAGGCACTGGCTC CCATGTCCTGGCCCTGAAGAGCCA TTCAGGGCCAGGACATGGGTGCCTTCAGCTCG Q148G GAAGGCACTGCATCTGGGTG GCTGTTCCATGTCCTGGCCACCCAGCCAGGACATGGAACAGC GATGCAGTGCCTTC K209A CCCCAAGAACTACCCCAAGGGTTGAACACGAAGCGCTTTTCCAT CAAAGATGGAAAAGCGCT CTTTGCCTTGGGGTAGTTCTTGGGTCGTGTTCAAC G

Production of IL-1β-Mutant: CCL20 Fusion Proteins.

IL-1β-CCL20 fusion proteins were produced in HEK293T cells. Forsmall-scale production, HEK293T cells were seeded in 6-well plates at400000 cells/well in DMEM supplemented with 10% FCS. After 24 hours,culture medium was replaced by medium with reduced serum (DMEM/5% FCS)and cells were transfected using linear PEI. Briefly, PEI transfectionmix was prepared by combining 1 μg expression vector with 5 μg PEI in160 μl DMEM, incubated for 10 minutes at RT and added to the wellsdropwise. After 24 hours, transfected cells were washed with DMEM andlayered with 1.5 ml OptiMem/well for protein production. Conditionedmedia were recuperated after 48 hours, filtered through 0.45μ filtersand stored at −20° C. IL-1β content in the conditioned media wasdetermined by ELISA according to the manufacturer's instructions (R&DSystems).

Cloning of the scTNF/Leptin Fusion Proteins.

The coding sequences of the wild-type (WT), L86S and L86N leptin weresynthesized by PCR from pMet7 plasmids expressing WT Leptin, Leptin L86Sor Leptin L86N, respectively, using the following primers:

forward 5′-GCAGATCTGTCGACATCCAGAAAGTCCAGGATGACACC-3′, reverse5′-CGATGCGGCCGCACATTCAGGGCTAACATCCAACTGT-3′.

This introduces a BgIII and a NotI site at the amino and carboxyterminus, respectively, of the leptin coding sequence. The PCR productwas digested with BgIII and NotI and cloned into pMET7-SIgK-HA-scTNFWT-6xGGS-FLAG (WT scTNF was generated by gene synthesis, GeneArt) openedwith BgIII and NotI, which reside in between the 6xGGS and FLAG. Thisgenerated pMET7-SIgK-HA-scTNF WT-6xGGS-mLeptin-FLAG, pMET7-SIgK-HA-scTNFWT-6xGGS-m Leptin L86S-FLAG and pMET7-SIgK-HA-scTNF WT-6xGGS-mLeptinL86N-FLAG.

Production of the scTNF/Leptin Fusion Proteins.

HekT cells were transfected with the different fusion protein constructsusing the standard calcium phosphate precipitation method. 48 hoursafter the transfection culture mediums were harvested and stored at −20°C. The concentration was determined with a commercial hTNFα ELISA(DY210, R&D systems).

Cell Lines

Hek 293T, HL116 and LL171 cell line were grown in DMEM supplemented with10% FCS.

Ba/F3-mLR and Ba/F3-mLR-TNFR1ΔCyt cells were maintained in RPMIsupplemented with 10% heat-inactivated FCS and 100 ng/ml leptin.

Assays

Phospho STAT1 Assay.

Single-cell suspensions were prepared from spleens isolated from C57Bl/6mice. Erythrocytes were depleted using red blood cell lysis buffer(Lonza). Splenocytes were treated for 30 min with mouse IFNα/β orXCL1-IFNα2-Q124R fusion protein in RPMI 5% fetal calf serum at 37° C.and then labelled with the BD Phosflow PE mouse anti-STAT1 (pY701)together with the Alexa Fluor 488-labelled anti-mouse CD11c (eBioscience#53-0114-80) and APC-labelled anti mouse CD8a (BD Bioscience #553035) oranti-mouse CD11c and Alexa 488-labelled anti-mouse CD8a according to BDBiosciences instructions. FACS data were acquired using a BD FACS Cantoand analyzed using either Diva (BD Biosciences) software.

NF-κB Reportergene Assay.

To assess IL-1R activation, we used HEK-Blue™ IL-1β cells that stablyexpress the IL-1R (Invivogen) and transfected them transiently with anNF-κB luciferase reportergene. Briefly, HEK-Blue™ IL-1β cells wereseeded in culture medium (DMEM/10% FCS) in 96-well plates (10000cells/well) and transfected the next day using the calciumphosphateprecipitation method with the indicated amounts of expression plasmidsand 5 ng/well of the 3 κB-Luc reportergene plasmid (Vanden Berghe etal., 1998). 24 hours post-transfection, culture medium was replaced bystarvation medium (DMEM) and 48 hours post-transfection, cells wereinduced for 6 hours with IL1-CCL20 fusion proteins. After induction,cells were lysed and luciferase activity in lysates was determined usingthe Promega Firefly Luciferase Assay System on a Berthold centro LB960luminometer.

Cell Proliferation Assay.

The Ba/F3-mLR cell line was generated by electroporation of Ba/F3 cellswith the pMet7-mLR vector. Stably expressing cells were selected bygrowing them on leptin instead of IL-3. Indeed, growth of Ba/F3 cells isdependent on IL-3, but when they express mLR, they also proliferate withleptin. To obtain the Ba/F3-mLR-TNFR1ΔCyt cell line, Ba/F3-mLR cellswere co-transfected with pMet7-HA-hTNFR1ΔCyt and pIRESpuro2 (Clontech)followed by puromycin selection and FACS sorting ofhTNFR1ΔCyt-expressing cells.

To assess cell proliferation, Ba/F3-mLR and Ba/F3-mLR-TNFR1ΔCyt cellswere washed, seeded in RPMI/10% iFCS in 96-well plates (10.000cells/well) and stimulated with the indicated amounts of leptin orfusion proteins. Four days later, 50 ul XTT (XTT Cell Proliferation KitII, Roche, 11 465 015 001) was added and incubated for 4 hrs beforemeasuring absorbance at 450 nm.

Example 1: IFN Activity of the XCL1/IFNα2-Q124R Fusion Protein isRestored on Cells Expressing XCR1

Mouse splenocytes were treated for 30 minutes with 1 nM XCL1-IFNα2-0124Ror with 10000 units/ml mouse IFNα/β. Cells were then fixed,permeabilized and stained with an anti-phospho STAT1 (PE), anti CD11c(Alexa Fluor 488) and anti CD8α (APC) and analyzed by FACS. FIG. 2 showsthat mouse IFN α/β induced STAT1 phosphorylation in all splenocytesubsets analysed. In contrast the XCL1-IFNα2-Q124R fusion proteininduced an IFN response only in the majority of cells belonging to theCD11c⁺ CD8α⁺ subset and in a minority of cells belonging to the CD11c⁺CD8α⁻ subset. The distribution of the splenocyte subsets responding tothe XCL1-IFNα2-Q124R fusion protein matches perfectly the expecteddistribution of XCR1, the XCL1 receptor (Dorner et al. 2009).

Example 2: IL1β Activity is Restored on Cells Expressing CCR6

HEK-Blue™ IL-1β cells, which stably express the IL-1R, were transientlytransfected with an NF-κB reportergene plasmid (5 ng/well) and an emptyvector or hCCR6 expression plasmid (10 ng/well). Mock- andCCR6-transfected cells were next treated for 6 hours with wild type ormutant IL1β-CCL20 fusion proteins (25 ng/ml), after which cells werelysed and NF-κB reportergene activity was determined. As evident fromFIG. 4A, cells expressing CCR6 responded with increased NF-κBreportergene activity to all investigated mutant IL1β-CCL20 fusionproteins as compared to mock-transfected cells. To evaluate the effectof the IL-1β-Q148G mutant, for which the targeting effect was mostapparent, in more detail, mock-transfected or CCR6-expressing HEK-Blue™IL-1β cells were treated for 6 hours with increasing doses of WT IL-1βor IL-1βQ148G-CCL20 fusion protein. FIG. 4B demonstrates thatoverexpression of CCR6 increased the activity of the WT IL-1β-CCL20fusion, but had a stronger potentiating effect for the IL-1βQ148G-CCL20fusion. The targeting effect was most prominent when IL1-β-CCL20 wasapplied to the cells at 12.5 ng/ml (FIG. 4C).

Example 3: Leptin Activity is Restored on Cells Expressing the TNFR

The proliferation of Ba/F3-mLR and Ba/F3-mLR-TNFR1ΔCyt cells after 4days of stimulation with the indicated amounts of leptin or theleptin-scTNF fusion proteins was assessed. As shown in FIG. 6A, bothcell lines do not proliferate in growth medium supplemented only withheat-inactivated serum. Moreover, the ability of leptin to induce Ba/F3proliferation is reduced when it is coupled to scTNF. Mutating L86within WT leptin into either a serine (L86S) or an asparagine (L86N)results in a moderate or a strong reduction of the affinity towards themouse leptin receptor, respectively. This reduction in affinitytranslates in a 3 versus 10 times less potent induction of proliferationof Ba/F3-mLR cells for leptin L86S versus L86N, respectively. Additionaltransfection of Ba/F3-mLR cells with the human TNF-R1 lacking itsintracellular domain (hTNFR1ΔCyt) introduces a non-functional receptor,which can function as a membrane bound extracellular marker. Clearly,the proliferative response upon stimulation with the L86S and L86Nleptin mutants coupled to scTNF is completely restored in Ba/F3-mLRcells that express the hTNFR1ΔCyt (FIG. 6B).

Example 4: In Vivo Targeting of an XCR1 Expressing Cell Population

According to Bachem et al. (Frontiers in Immunology 3, 1-12. 2012), XCR1expressing cells represent the major part of CD11c+CD8α+ spleen cellpopulation and a minor part of CD11c+ CD8α− spleen cell population.C57Bl/6 mice were injected iv with the indicated amount ofXCL1-IFNα2-Q124R or with 1 000 000 units of natural murine IFNα/β orPBS. After 45 min, spleen cells were analyzed by FACS for P-STAT1 in thefollowing cell population: CD11c− CD8α−, CD11c− CD8α+, CD11c+CD8α+,CD11c+CD8α−. The results are shown in FIG. 7. From these results, it isclear that the fusion construct can target and induce a response in aminor fraction of the population (about 0.1% of the total cells),whereas the IFN sensitive cells that do not express the marker are notaffected. Indeed, wild type IFN is also affecting the CD11c+ CD8α−cells, whereas those cells are not affected by the fusion construct,clearly proving the specific action of the fusion.

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1. A composition comprising a fusion protein comprising at least twocytokines, wherein: at least one cytokine comprises a mutation thatstrongly reduces binding activity to its receptor, and at least onecytokine is a wild-type cytokine which provides cell-specific targetingthat restores activity of the mutant cytokine on the targeted cells. 2.The fusion protein according to claim 1, further comprising a GGSlinker.
 3. The composition according to claim 1, wherein said cytokinesare XCL1 and IFNα2.
 4. The composition according to claim 1, whereinsaid cytokines are CCL20 and IL-1β.
 5. The composition according toclaim 1, wherein said cytokines are TNF and leptin. 6.-8. (canceled) 9.The composition according to claim 3, wherein the IFNα2 comprises themutation.
 10. The composition according to claim 9, wherein the XCL1 isnot mutated.
 11. The composition according to claim 4, wherein the IL-1βcomprises the mutation.
 12. The composition of claim 11, wherein theIL-1β mutation is at a position selected from 120, 131, 146, 148, and209.
 13. The composition of claim 12, wherein the IL-1β mutation isselected from R120G, Q131G, H146A, Q148G, and K209A.
 14. The compositionaccording to claim 11, wherein the CCL20 is not mutated.
 15. Thecomposition according to claim 5, wherein the leptin comprises themutation.
 16. The composition of claim 15, wherein the leptin mutationis at position
 86. 17. The composition of claim 16, wherein the leptinmutation is selected from L86S and L86N.
 18. The composition accordingto claim 15, wherein the TNF is not mutated.
 19. A method of stimulatingan immune response in a cell, comprising contacting the cell with acomposition comprising a fusion protein comprising at least twocytokines, wherein: at least one of the cytokines has a mutation whichprovides strongly reduced affinity for its receptor as compared to wildtype cytokine.
 20. The method of claim 19, wherein NFκB is activated.21. The method of claim 20, wherein the mutated cytokine is a humanIL-1β mutant comprising a mutation selected from R120G, Q131G, H146A,Q148G, and K209A.
 22. The method of claim 21, wherein one of thecytokines is wild type CCL20.